DESCRIPTION Calcium signals have important roles in many neuronal processes, including the release of neurotransmitter, gene expression as well as different aspects of neuronal plasticity. However, the mechanisms by which different intracellular calcium release mechanisms and different calcium influx pathways can selectively activate down-stream targets are not yet understood. Localization of calcium signals and the localized activation of distinct calcium effectors are thought to be important means through which specificity can be achieved. The objective of this grant is to define, in living cells, to which extent calcium signals are localized and to determine how localized calcium signals are used for the specific activation of calmodulin and of Ca2+/calmodulin-dependent protein kinase II (CaMkinase). A better understanding of the pleiotropic functions of calcium signals is of fundamental importance for a large number of neuronal diseases. During the last funding period, the applicants have developed several new fluorescent methods to attack their objective. In particular, they have developed an efficient"microporation" technique to introduce RNA and other macromolecules into different neuronal and non-neuronal cell models. RNA transfection has significant advantages over DNA transfection for the rapid and efficient expression of GFP-tagged proteins in different cell types. The applicants will study the local control of depolarization-induced calcium influx and local IP3-gated calcium release by using newly developed localized calcium indicators, localized markers for calcium stores as well as GFP-based fluorescent probes to measure calmodulin binding. Specifically, they will investigate: 1. whether localized calcium signals are an essential part of calcium-mediated signal transduction processes, 2. whether calmodulin is a local mediator of signaling transduction and 3. which mechanism is responsible for the localization of CaMkinase to different cellular sites. The results from these experiments will be integrated into a model of localized neuronal signal transduction mediated by calcium, calmodulin and CaMkinase.